Solid state synthesis is applicable to the preparation of a wide variety of polymeric compounds such as nucleobase-containing polymers (for example oligonucleotides and their analogs), amino acid containing polymers (for example proteins, peptides, and their analogs). Solid state synthesis also is applicable to the preparation of compounds by combinatorial methods.
Oligonucleotides have been used in various biological and biochemical applications. They have been used as primers and probes for the polymerase chain reaction (PCR), as antisense agents used in target validation, drug discovery and development, as ribozymes, as aptamers, and as general stimulators of the immune system. As the popularity of oligonucleotides has increased, the need for producing greater sized batches, and greater numbers of small-sized batches, has increased at pace. Additionally, there has been an increasing emphasis on reducing the costs of oligonucleotide synthesis, and on improving the purity and increasing the yield of oligonucleotide products.
A number of innovations have been introduced to the art of oligonucleotide synthesis. Amongst these innovations has been the development of excellent orthogonal protecting groups, activators, reagents, and synthetic conditions. The oligonucleotides themselves have been subject to a variety of modifications and improvements. Amongst these are chemistries that improve the affinity of an oligonucleotide for a specific target, that improve the stability of an oligonucleotide in vivo, that enhance the pharmacokinetic (PK) and toxicological (Tox) properties of an oligonucleotide, etc. These novel chemistries generally involve a chemical modification to one or more of the constituent parts of the oligonucleotide.
The term “oligonucleotide” thus embraces a class of compounds that include naturally-occurring, as well as modified, oligonucleotides. Both naturally-occurring and modified oligonucleotides have proven useful in a variety of settings, and both may be made by similar processes, with appropriate modifications made to account for the specific modifications adopted. A naturally occurring oligonucleotide, i.e. a short strand of DNA or RNA may be envisioned as being a member of the following generic formulas, denominated oligo-RNA and oligo-DNA, respectively, below:
wherein m is an integer of from 1 to about 100, and Bx is one of the naturally occurring nucleobases.
Physiologic pH, an oligonucleotide occurs as the anion, as the phosphate easily dissociates at neutral pH, and an oligonucleotide will generally occur in solid phase, whether amorphous or crystalline, as a salt. Thus, unless otherwise modified, the term “oligonucleotide” encompasses each of the anionic, salt and free acid forms above.
In essence, a naturally occurring oligonucleotide may be thought of as being an oligomer of m monomeric subunits represented by the following nucleotides:
wherein each Bx is a nucleobase, wherein the last residue is a nucleoside (i.e. a nucleotide without the 3′-phosphate group).
As mentioned above, various chemistry modifications have been made to oligonucleotides, in order to improve their affinity, stability, PK, Tox, and other properties. In general, the term oligonucleotide, as now used in the art, encompasses inter alia compounds of the formula:
wherein m is an integer from 1 to about 100, each G1 is O or S, each G2 is OH or SH, each G3 is O, S, CH2, or NH, each G5 is a divalent moiety such as O, S, CH2, CFH, CF2, —CH═CH—, etc., each R2′ is H, OH, O-rg, wherein rg is a removable protecting group, a 2′-substituent, or together with R4′ forms a bridge, each R3′ is H, a substituent, or together with R4′ forms a bridge, each R4′ is H, a substituent, together with R2′ forms a bridge, together with R3′ forms a bridge, or together with R5′ forms a bridge, each q is 0 or 1, each R5′ is H, a substituent, or together with R4′ forms a bridge, each G6 is O, S, CH2 or NH, and each G7 is H, PO3H2, or a conjugate group, and each Bx is a nucleobase, as described herein (i.e. naturally occurring or modified).
The standard synthetic methods for oligonucleotides include the solid phase methods first described by Caruthers et al. (See, for example, U.S. Pat. No. 5,750,666, incorporated herein by reference, especially columns 3-58, wherein starting materials and general methods of making oligonucleotides, and especially phosphorothioate oligonucleotides, are disclosed, which parts are specifically incorporated herein by reference.) These methods were later improved upon by Köster et al. (See, for example, U.S. Pat. No. RE 34,069, which is incorporated herein by reference, especially columns, wherein are disclosed, which parts are specifically incorporated herein by reference.) These methods have further been improved upon by various inventors, as discussed in more detail below. Methods of synthesizing RNA are disclosed in, inter alia, U.S. Pat. Nos. 6,111,086, 6,008,400, and 5,889,136, each of which is incorporated herein in its entirety. Especially relevant are columns 7-20 of U.S. Pat. No. 6,008,400, which are expressly incorporated herein by reference.
The general process for manufacture of an oligonucleotide by the Köster et al. method may be described as follows:
First, a synthesis support is prepared by covalently linking a suitable nucleoside to a solid support medium (SS) through a linker. Such a synthesis support is as follows:
wherein SS is the solid support medium, LL is a linking group that links the nucleoside to the support via G3. The linking group is generally a di-functional group, which covalently binds the ultimate 3′-nucleoside (and thus the nascent oligonucleotide) to the solid support medium during synthesis, but which is cleaved under conditions orthogonal to the conditions under which the 5′-protecting group, and if applicable any 2′-protecting group, are removed. T′ is a removable protecting group, and the remaining variables have already been defined, and are described in more detail herein. Suitable synthesis supports may be acquired from Amersham Biosciences under the brand name Primer Support 200™. The solid support medium having the synthesis support attached thereto may then be swelled in a suitable solvent, e.g. acetonitrile, and introduced into a column of a suitable solid phase synthesis instrument, such as one of the synthesizers available form Amersham Biosciences, such as an ÄKTAoligopilot™, or OligoProcess™ brand DNA/RNA synthesizer.
In the foregoing method, synthesis is carried out from 3′- to 5′-end of the oligomer. In each cycle, the following steps are carried out: (1) removal of T′, (2) coupling, (3) oxidation, (4) capping. Each of the steps (1)-(4) may be, and generally is, followed by one or more wash steps, whereby a clean solvent is introduced to the column to wash soluble materials from the column, push reagents and/or activators through the column, or both. The steps (1)-(4) are depicted below:

In general, T′ is selected to be removable under conditions orthogonal to those used to cleave the oligonucleotide from the solid support medium at the end of synthesis, as well as those used to remove other protecting groups used during synthesis. An art-recognized protecting group for oligonucleotide synthesis is DMT (4,4′-dimethoxytrityl). The DMT group is especially useful as it is removable under weakly acid conditions. Thus, an acceptable removal reagent is 3% DCA in a suitable solvent, such as acetonitrile. The wash solvent, if used, may conveniently be acetonitrile.
The support typically is a controlled pore glass or a polymeric bead support. Some polymeric supports are disclosed in the following patents: U.S. Pat. No. 6,016,895; U.S. Pat. No. 6,043,353; U.S. Pat. No. 5,391,667 and U.S. Pat. No. 6,300,486, each of which is specifically incorporated herein by reference.
After removal of protecting group T′, the next step of the synthetic cycle is the coupling of the next nucleoside synthon. This is accomplished by reacting the deprotected support bound nucleoside with a nucleoside phosphoramidite, in the presence of an activator, as shown below:

The amidite has the structure:
wherein pg is a phosphorus protecting group, such as a cyanoethyl group, and NRN1RN2 is an amine leaving group, such as diisopropyl amino. See, Köster et al., supra, for information on manufacturing of the amidite. Typically used activators include for example tetrazole, dicyano imidazole, or pyridinium salts. Other suitable amidites, and methods of manufacturing amidites, are set forth in the following patents: U.S. Pat. No. 6,133,438; U.S. Pat. No. 5,646,265; U.S. Pat. No. 6,124,450; U.S. Pat. No. 5,847,106; U.S. Pat. No. 6,001,982; U.S. Pat. No. 5,705,621; U.S. Pat. No. 5,955,600; U.S. Pat. No. 6,160,152; U.S. Pat. No. 6,335,439; U.S. Pat. No. 6,274,725; U.S. Pat. No. 6,329,519, each of which is specifically incorporated herein by reference, especially as they relate to manufacture of amidites. Suitable activators are set forth in the Caruther et al. patent and in the Köster et al. patent. Especially suitable activators are set forth in the following patents: U.S. Pat. No. 6,031,092 and U.S. Pat. No. 6,476,216, each of which is expressly incorporated herein by reference.
The next step of the synthesis cycle is oxidation, which indicates that the P(III) species is oxidized to a P(V) oxidation state with a suitable oxidant:
wherein G1 is O or S.
The oxidant is an oxidizing agent suitable for introducing G1. In the case where G1 is oxygen, a suitable oxidant is set forth in the Caruthers et al. patent, above. In cases where G2 is sulfur, the oxidant may also be referred to as a thiation agent or a sulfur-transfer reagent. Suitable thiation agents include the so-called Beaucage reagent, 3H-1,2-benzothiol, phenylacetyl disulfide (also referred to as PADS; see, for example the patents: U.S. Pat. Nos. 6,114,519 and 6,242,591, each of which is incorporated herein by reference) and thiouram disulfides (e.g. N,N,N′,N′-tetramethylthiouram disulfide, disclosed by U.S. Pat. No. 5,166,387). The wash may be a suitable solvent, such as acetonitrile.
The oxidation step is followed by a capping step, which although not illustrated herein, is an important step for synthesis, as it causes free 5′-OH groups, which did not undergo coupling in step 1, to be blocked from being coupled in subsequent synthetic cycles. Suitable capping reagents are set forth in Caruthers et al., Köster et al., and other patents described herein. Suitable capping reagents include a combination of acetic anhydride and N-methylimidazole.
Synthetic cycle steps (1)-(4) are repeated (if so desired) n−1 times to produce a support-bound oligonucleotide:
wherein each of the variables is as herein defined.
In general, the protecting group pg may be removed by a method as described by Caruthers et al. or Köster et al., supra. Where pg is a cyanoethyl group, the methodology of Köster et al., e.g. reaction with a basic solution, is generally suitable for removal of the phosphorus protecting group. In some cases it is desirable to avoid formation of adducts such as the N1-cyanoethyl thymidine group. In these cases, it is desirable to include in the reagent a tertiary amine, such as triethylamine (TEA) as taught in U.S. Pat. No. 6,465,628, which is expressly incorporated herein by reference. In general, where the nucleobases are protected, they are deprotected under basic conditions. The deprotected oligonucleotide is cleaved from the support to give the following 5′-protected oligonucleotide:
which may then be purified by reverse phase liquid chromatography, deprotected at the 5′-end in acetic acid, desalted, lyophilized or otherwise dried, and stored in an inert atmosphere until needed. Optionally, the G3H group may be derivatized with a conjugate group. The resulting oligonucleotide may be visualized as having the formula:

While synthesis on a solid phase support medium is known, there is a need for a solid support medium that has improved properties, especially with respect to loading (expressed in mmol of first nucleoside bound to the solid support medium per gram of solid support medium, or simply mmol/g), consistent swelling properties during the various synthesis cycles, and quality of full length oligomer produced during synthesis. Additionally, the support should be facile to manufacture. This invention is directed to these, as well as other, important ends.